Kidney failure is a major cause of morbidity and mortality in the United States and worldwide. One of the major factors that influences the progression of kidney failure is the amount of proteinuria. Our long term goal is to define the precise mechanisms of proteinuria and glomerular disease, so as to develop new treatments in the future. The overall goal of this proposal is to understand the role of transcriptional factor Zinc Fingers and Homeoboxes 3 (ZHX3) in the pathogenesis of proteinuria. In Specific Aim 1, we will study the post-translational cleavage and phosphorylation of ZHX3 prior to migration into the nucleus. We will then study whether blocking nuclear import or export of ZHX3 causes alterations in the gene expression profile of ZHX3 target genes. In Specific Aim 2, the individual effects of both ZHX3 protein fragments on the gene expression profile in cultured cells will be studied by real time PCR. Next, interaction with other ZHX family members and other known and putative interacting protein will be assessed. Specific factors that influence the binding of ZHX3 to the Cathepsin L and podocalyxin promoters will be studied by a combination of chromatin immunoprecipitation and site directed mutagenesis. Next, the individual and combined influences of the the various ZHX family members and their known interacting partners on the promoter activity of selected genes will be assesed.ln Specific Aim 3, a podocyte-specific conditional ZHX3 knockout mouse will be generated to study the role of ZHX3 in normal podocyte function. Selected parts of the mouse ZHX3 gene will be cloned into the targeting vector, which will be electroporated into mouse embryonic stem cells. After screening for homologous recombination, FLP recombinase will be used to remove the neomycin resistance cassette, the targeted stem cells fused with eight stage embryos, and chimeric mice generated. After further breeding and genotyping, the floxed ZHX3 mouse will mated with a podocin Cre mouse, to generate, after several cycles of breeding and genotyping, ZHX3 -/- and +/- mice. Urinary protein excretion of the ZHX3 deficient mice will be assessed, and detailed morphological characterization undertaken. Studying the development of kidney disease and the leakage of protein in the urine in rodents will help us better understand the process of kidney disease in humans. This will lead to the devlopment of appropriate treatment strategies in the future.